Field study on buffaloe oedematous skin disease in Assiut Gavanorate: a model study.

Animal Health Research Institute (AHRI).

Assiut Lab.

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Field study on buffaloe oedematous skin disease in Assiut Gavanorate:

a model study.

دراسة حقلية لمرضالتهاب الجلد الاوديمى فى الجاموس فى محافظة أسيوط : نموذج دراسة.

سيد محمد سيد , حسن زکى راتب* , محسن ابراهيم عرفه, محمد محمد عبد الحفيظ, أحمد عبد الفتاح عامر*

Sayed. S.M.; Rateb.H.Z. *; Arafa. M.I.; Abdel-Hafeez.M.M.; and Aamer.A.A. *

* Dep. of Ani. Med. Faculty of  Vet. Med. Ass. Univ.

الملخص العربي

         نظراً لظهور مرض الجلد الأوديمي في الجاموس في صورة متکررة وأصبح متوطن في مصر هدف البحث إلي دراسة حقلية للمرض في منطقة محدودة. واشتملت الدراسة علي 44 جاموسة مصابة بمرض الجلد الأوديمي. لوحظ شکلين للصورة الإکلينيکية للمرض: الأوديمي (95.5%) والعقدي (4.5%) بينما لم تسجل أي حالات للصورة المتقرحة. وقد کانت الأجزاء الأمامية للجسم أکثر عرضة للإصابة (79.5%). تم عزل 35 عترة من الميکروب المسبب للمرض (کوريني سودوتيبرکلوزيس ) وقد کان في صورة منفردة في 32 حالة (80 %) وفي صورة مشترکة مع العنقودي إبيدرميدس في 3 حالات (7.5%) .

أظهرت 24 عترة (68.6%) نتائج إيجابية لاختبار اختزال النيترات و کانت من النوع سيروتيب I بينما الإحدى عشر عترات الأخرى(31.4%) کانت من نوع سيروتيب II . کلا النوعين تم عزلهما من ذبابة الهيبوبوسيکا إکوينا بينما تم عزل  سيروتيب I من کل من الشرنقة والجيل الثاني من الذبابة السابق ذکرها.

وقد اثبتت اهذه الدراسة ان ذبابة الهيبوبوسيکا اکوينا هى المسؤل عن نقل هذا المرض حيث تم عزل الميکروب المسبب له من کل من السطح الخارجى للذبابة والمحتويات الداخلية لها. وکذلک من الشرنقة والجيل الثانى للحشرة والذى تم تربيته معمليا بينما لم يتم عزل هذا الميکروب من القمل الماص للدم والموجود على الجاموس المصاب بالمرض . هذا يدل على ان طبيعة التعايش الداخلى لميکروب الکوريني سودوتيبرکلوزيس قاصراعلى ذبابة الهيبوبوسيکا اکوينا .

واظهرت نتائج اختبار الحساسية للمضادات الحيوية ان جميع العترات المعزولة اعطت 100% ايجابية لکل من التوبراميسين والجنتاميسين والسيبروفلوکساسين, يليها الاوکسيتتراسيکلين بنسبة 84%. جميع العترات المعزولة اظهرت مقاومة لکل من البنسيلين و الامبسيلين والکلوکساسيلين.

وبالعلاج باستخدام المضادات الحيوية طبقا لاختبارات الحساسية وحقن مضادات الهستامين فضلا عن مکافحة الطفيليات الخارجية امکن الوصول الى نسب شفاء 97.7%.

Summary

            As the buffaloe oedematous skin disease (OSD) became an endemic disease in Egypt, the present investigation objected to approach the subject in a localized district as a field study. Through a village clinic, 44 buffaloe cows suffering from OSD were included in the study. The disease was observed in two clinical forms, oedematous (95.5 %) and nodular (4.5 %), where ulcerative form was not recorded. Anterior parts of the body were mostly affected (79.5 %). Corynebacterium pseudotuberculosis was the causative agent of the disease. It was isolated from

 aspirated exudates as single infection from 32 (80.0%) and as mixed infection with Staphylococcus epidermidis from 3 (7.5 %). Twenty four (68.6 %) of these isolates showed nitrate reduction positive reactions (serotype I), while the other 11 strains  

(31.4 %) were nitrate reduction negative (serotype II). Both serotypes were recovered from adult Hippobosca equina flies. Strains of serotype I were isolated from either pupae or laboratory developed fly.

          The present study proved the sole role of H.equina fly in disease transmission. C.pseudotuberculosis was isolated from external body surface, internal body content of the fly, pupae and the second generation. Failure of its isolation from blood sucking lice confirmed that the endosymbiotic nature of C.pseudotuberculosis was limited to H.equina fly.

          Antibiogram of the isolated bacteria revealed their sensitivity 100 % for Tobrarmycin, Gentamycin and Ciprofluxacin followed by Oxytetracycline (84 %). All bacterial isolates showed resistance against Penicillin, Ampicillin and Cloxacillin. Treatment regimen basing on antibiotic, antihistaminic administration and ectoparasitic eradication achieved recovery rate of 97.72 %.

Introduction

   Oedematus skin disease (OSD) in buffaloes nowadays becomes an endemic disease in Egypt (Selim, 2001). It was fully studied and discussed through several investigations in different governorates which established that  the etiological agent was Corynebacterium pseudotuberculosis (Soliman et.al.,1963; Fouad et.al.,1972; Ibrahim et.al.,1983; Barakat et.al.,1984; Esmat,1984; Mostafa,1984; Hassan,1988; Zaghawa and El-Gharib,1996; Ghoneim et.al.,1999 and Ghoneim et.al., 2001 and others). However reports on occurrence of the disease in Assiut governorate still apparently brief (khalel et.al.,1995; Ali & Zaitoun, 1999 and Sayed, 2001). Antigenically C.pseudo. has two serotypes. Serotype I causes caseous lymphadenitis in sheep, while serotype II causes abscesses in horse and OSD in buffaloes (Barakat et.al.,1985). Both serotypes may infect cattle (Yeruham et.al.,1997 and Yeruham et.al., 2003). Oedematous skin disease has 3 clinical forms; oedematous, nodular and ulcerative (Al-Gaabry & Ammar, 1999).

         Several authors suggested that the route of transmission of buffaloe Oedematous Skin Disease is through the mechanical way only either by contamination of external environment (soil & water) with C. pseudo. or by external parasites (Khater et.al., 1983; Barakat et.al., 1984; Khalil et al., 1995; Sayed, 2001 and Spier et.al, 2004). However, the entry of the host into cattle is not well documented (Yeruham et.al., 2004).

   The disease appeared in some villages of Assiut governorate as an epidemic every 3-4 years. During May-August 2006 cases were recorded in El-Fayama village 15 km East Assiut city.

         The aim of the present study is to record the occurrence, clinical manifestations and the role of ectoparasites in transmission of disease with trials for treatment of affected cases as a field study.

Material and Methods

         Animals: Through a private clinic in Fayama village – previously mentioned – the study conducted on 44 buffaloes cows (11 pregnant and 33 non pregnant) aging 3-9 years showing oedematus skin lesions during the period May – August.2006.

Bacteriological sample collection: Lesion form, its body distribution and physical appearance of its aspirated exudates was recorded and tabulated. Forty three closed and one opened – which spontaneously closed – localized or diffuse, cutaneous or lymph node were observed. Every lesion was aspirated aseptically for bacterial isolation. Aspirated exudates specimens were inoculated into nutrient broth media overnight and incubated aerobically at 37ºC, then were streaked onto 10% sheep blood agar (24 - 48 hrs`).

   Growing colonies were purified and identified morphologically by Gram's stain. Biochemically tested for motility, glucose and maltose fermentation, catalase activity and nitrate reduction were adopted according to Quinn et.al., (1994).

For the parasitological investigation: Forty adult flies and 30 lice were collected directly by hand or using forceps from infected buffaloes. They were kept into plastic sacs or wide-naked bottles for laboratory examination where they taxonomically identified under binocular microscope according to their morphological characters (Soulsby, 1982 and Kettle, 1984).

       Some collected flies were still alive for 24 hours until deposited their larvae (Full mature larvae) inside the collected sacs, larvae pupated rapidly. The achieved pupae were incubated at room temperature in plastic containers containing sand and covered with a piece of gauze until giving adult fly (Baraka, 1983). Different stages of collected external parasites were measured and photomicrographed.

Bacterialogical examination of Blood sucking ectoparasites: In parallel to samples for bacteriological study twenty two flies and were gathered in sterile test tubes from infested animals (17 affected buffaloes, 4 cattles and a donkey) in addition to one laboratory developed fly for bacterial existence was also examined as follow: fly was inoculated as it is into sterile nutrient broth tube for isolation of body surface, legs and external mouth parts contamination. Then a forceps catched fly was washed several times using sterile distilled water. The washed fly was crushed, destructed and macerated into another nutrient broth tube for isolation of gut bacterial content. Also twenty lice from only affected buffaloes were managed in the same technique to obtain their external and internal bacterial contents.

    From the laboratory deposited pupae, 2 were burst into nutrient broth to isolate their bacterial contents.

In vitro antimicrobial susceptibility testing: of the lesion isolated strains was carried out according to Bauer et al, (1966) using disc diffusion technique against: Amekin (30 µg), Ampicillin (10 µg),  Cloxacillin (10 µg),  Ciprofluxacin (5µg),  Ggentamycin (10 µg), Oxytetracycline (30 µg) penicillin (10 ΙU),  Streptomycin (10 µg) and Tobramycin (10 µg). [Bioanalyse]

   Treatment regimens were applied as the cases indicated.

Results  

        The obtained bacteriological results were tabulated in tables 1-5 and plates 1& 2.

Parasitological findings:

         In the present study, all diseased buffaloes were infested with dark leathery flies identified as Hippobosca equina. Adult flies were more abundant on stabled diseased animals. They mainly aggregated under the tail, on the udder, around genitalia and inner aspect of thighs. Flies were dark brown in color measuring 9 x 4.5- 10.5 x 5.0 mm. Their abdominal segmentation was indistinct. Wings were longer than body length while wing veins crowded towards the anterior border. Flies had three pair of feets provided with strong claws (plate 3 fig: 1).

Out of 8 full mature larva, 5 pupated (6-10) hours. The larva is creamy in color, oval shape measuring 1.5 x 2.5 mm. It was provided with a small spine posteriorly (plate 3 fig: 2). Pupation period was 30 days where the pupa is broadly oval with two round postero-lateral spiracular lobes. Pupa was yellowish in color measuring 4.0 x 2.5- 4.5 x 3 mm. It was soft and covered with sticky layer but at 24 hours later, it became dark red to black in color and quit hard (plate3 fig: 3).

 Five diseased animals were infested also with sucking lice identified as Haematopinus eurysternus. The sucking louse has a relatively short head and broad thorax and abdomen. Its measuring was 4 x 2 – 5 x 2.5 mm. (plate3 fig: 4).

Discussion

Retrospectively, OSD appeared approximately in epidemic cycle with an interval of three years during which the disease appeared sporadically (Al-Gaebary and Ammar, 1999). Barakat et.al., (1985) recorded five outbreaks of bovine OSD in Egypt mostly in buffaloes. Analysis of previous data revealed that the seasonal pattern of the disease begins as sporadic cases reaching the maximum in summer months (May – July) followed by gradual receding in winter (Fouad et.al., 1974).

As mentioned in the above premise, the disease is caused by C.pseudotuberculosis, the dermonecrotic effect is attributed to its phospholipidase exotoxin- vascular toxin- with permeability factor (Afzal et al., 1996).

             Oedema begins as an inflammatory oedema up to surrounding tissue necrosis    (Selim, 2001). Consequently three clinical forms were recorded; oedematus, nodular and ulcerative (Al- Gaebary and Ammar, 1999).

           The present findings revealed that affected buffaloes showed forty two (95.5%) oedematus and two (4.5%) nodular forms (table 1). A lesion in multiple nodular case was ruptured seemed to be ulcerative form (plate 1, fig. d). Oedematus (diffuse) form may be only skin lesions involved. Associated lesion appeared also in prescapular lymph node (plate 1fig: a& b). Anterior parts of the body were mostly affected (79.5%) more than posterior ones (20.5%) (Table 1). Ali and Zaitoun (1999) achieved that lesions related to the anterior parts of the body resembled 80.7%.

       The site of OSD correlated with the feeding habitat of H. equina flies. Such flies attach themselves to the inner sides of limbs, belly and neck (Hafez and Hilali, 1978).

 C.pseudotuberculosis was isolated from 35 (87.4%) lesion samples (table2). Abou-Zaid and Hammam (1994), Khalil et.al., (1995), Zaghawa and El-Gharib (1996), Ali and Zaitoun (1999) and Sayed (2001) detected C. pseudotuberculosis in 100, 83.3, 88.2, 94.5 and 100% of the infected buffaloes, respectively.

It is well documented that C. pseudotuberculosis showed polymorphism of isolates recovered from different animal species allover the world (Brown and Olander, 1987; Claus and Rikisa, 1991 and Sutherland et al., 1996). C. pseudotuberculosis was grouped in two different biotypes (serotypes) which differ from each other biochemically, serologically and epidemiologically. The most common feature of variation is the ability of serotype I to reduce nitrate to nitrite and the failure of serotypeII to do that (Shpigel et al., 1993). Barakat et al., (1984) reported that serotype I infects only sheep and goats causing caseous lymphadenitis but not be known to infect any of other animals. Later on, Shpigel et al., (1993) isolated it from cutaneous lesion in cattle. Yeruham et al., (2003) isolated serotype II from cutaneous lesions in cattle. Both serotypes were isolated in the same time from cattle ulcerative dermatitis (Yeruham et al., 2004). So it is obvious that C.pseudotuberculosis serotypes are not host specific pathogens. Through the present study-biochemically- both C.pseudoyuberculosis serotypes were recovered from OSD lesions and H. equina flies (table3).

          Regarding disease transmission, all available literature suggested only mechanical way either by direct contact between uninfected animals with infected ones or contaminated soil or water sources (Zaghawa and El-Gharib, 1994; Khalil et al., 1995; Ali and Zaitoun, 1999 and Doherr et al., 1999).

Direct contact of the uninfected animals with contaminated external parasites (mechanically) either by Musca domestica or H. equina was stated by many authers (Hafez and Hilali, 1978; Addo, 1983; Abou-Zaid and Hammam, 1994; Sayed, 2001; Yeruham et al., 2003 and Spier et al., 2004).

These suggestions could be accepted when lesions (source of infection) were opened in case of ulcerative form, but these contaminations cannot invade intact skin unless the insects were blood suckling.

         In the present work, C. pseudotuberculosis strains were isolated from 11(50 %) animal hosted H. equina flies (table 2) after 24 hours post gathering, also from two pupae after 24-48 hours and finally from the lab developed fly 30 day post pupa incubation (vertical transmission).

The successful positive isolation from all H. equina developmental stages, while it could not be isolated from all lice already infested the diseased buffaloes - in spite of both insects have mouth parts adapted for sucking blood - explained the symbiotic association between C. pseudotuberculosis and H .equina. Achieved results declared the endosymbiotic nature of C. pseudotuberculosis is limited to H. equina fly only. On other hand all infected cases were detected in summer season which correlated with the peak of population of H. equina(Hafez, et al., 1979) in addition to failure of isolation of C. pseudotuberculosis from lice. All these factors are confirmed the sole role of H. equina as a main vector of transmission of OSD.

Sensitivity tests of isolated strains:

         The present antimicrobial susceptibility testing showed that all tested C.pseudotuberculosis strains were highly sensitive to tobramycin, gentamycin and ciprofloxacin (100%) then oxytetracyclin with 80% (table 4). High sensitivity of gentamycin followed by oxytetracyclin was recorded by Abou-Zaid and Hamam (1994), Khalil et al., (1995), Ali and Zaitoun (1999) and Sayed (2001).

        Treatment in the present investigation was designed on three bases; the firstly was the onset use of the antibiotic of choice based on antimicrobial susceptibility testing for five successive days. The secondly was suppression and oedema inhibition through I/M administration of corticosteroids. Finally Hippobosca equina control through S/C injection of all infected animals with ivermectin (table 5). All in parallel ways, all stabled animals were atomized externally with diazinone (1%) three successive times with ten days intervals to fight all parasite developmental stages. Stables walls particularly their cracks were atomized with 1% diazinone also three times as mentioned above.

    Through the present study, treatment of OSD was carried out as shown in (table 5). It was beneficial in case it act as enlarged suprascapular lymph node to be surgically excised in order to reduce the infection and their toxins which have dermonecrotic effect (plate2, fig a-c). Surgical interference was previously recommended by Khalil et al., (1995), but it be prohibited in early stages except in case of secondary invasion with pyogenic micro-organisms (Selim, 2001).

Owining to application of this treatment regimen, recovery rate achieved was 97.72 % (table 5) at three weeks post administration. Great success in treatment would be obtained when be designed to remove C.pseudotuberculosis from tissues using antibiotics and inhibit oedema by hydrocortisone ( Sahar, 1998 and Selim, 2001 ). Al-Gaabary and Ammar (1999) concluded recovery rate of 80 & 90% for the two treated groups three weeks but later reached 100 % for both by the fifth week.

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        (Table 1) body distribution and forms of skin lesions in buffaloes OSD.

(Table 2): Bacterial species isolated from skin lesion of OSD and blood sucking insects hosted.

H .e: Hippobosca equina               B.S: External body surface                                   I.B.C.: Internal body content

                                              (Table 3): Nitrate reduction (NR) test of C.pseudotuberculosis isolates.

(Table 4): Antimicrobial susceptibility testing of bacterial isolates recovered from OSD in buffaloes.

Tob: Tobramycin                                               CIP: Ciprofluxacin                                  Amp: Ampicillin   

Amk: Amekin                                                       S: Streptomycin                                         P: Penicillin

GN: Ggentamycin                                              CX: Cloxacillin                                     T: Oxytetracycline      

(Table 5):Treatment of buffaloes oedematous skin disease and recovery rate.

                              Ciprofloxacin: Cipro. I/M.    1cc/ 40 kg b.wt. 3-5 days.

                              Oxytetracycline: L.A terramycin deep I/M.  1cc /10 kg b.wt/ 48 hrs 2 doses..

                              Flunixine: finadyne, I/V 2 ml/50kg b. wt. (Schering-plough veterinarie- france).

                              Phenylbutazone: buta-fenil slow I/V, 1-2 gm / animal.  (laboratories jornel, S.A.- Mixico).

                              Diphenohydramine HCL: allergamine I/V 1mg/kg b.wt. (Aveco-jordan).

                              Fluid therapy: sodium hydroxide 0.9% slow I/V infusion (ADWIC-Egypt).

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